Table Of ContentMolecular Biology
Biochemistry and Biophysics
31
Editors:
A. Kleinze(ler, Philadelphia . G. F. Springer, Evanston
H. G. Wittmann, Berlin
Advisory Editors:
C. R. Cantor, New York . F. Cramer, Gottingen
F. Egami, Tokyo . M. Eigen, Gottingen . F. Gros, Paris
H. Guifreund, Bristol . B. Hess, Dortmund
H. lahrmiirker, Munich . R. W. leanloz, Boston
E. Katzir, Rehovot . B. Keil, Gif-sur-Yvette
M. Klingenberg, Munich . I M. Klotz, Evanston
W. T. 1. Morgan, SuttoniSurrey . K. Muhlethaler, Zurich
S. Ochoa, New York' G. Palmer, Houston
I Pecht, Rehovot . R. R. Porter, O:xford
W. Reichardt, Tubingen . R. A. Reisfeld, La Jolla
H. Tuppy, Vienna . 1. Waldstrom, Malmo
Membrane Spectroscopy
Edited by Ernst Grell
With Contributions by
D. F. Bocian S. I. Chan M. C. Foster N. P. Franks
U. P. Fringeli E. Grell Hs. H. Giinthard 1. Heesemann
y. K Levine M. M. Long R. C. Lord D. Marsh
R. Mendelsohn N. O. Petersen H. Ruf D. W. Urry
1. Yguerabide H. P. Zingsheim
With 146 Figures
Springer-Verlag
Berlin Heidelberg New York 1981
Dr. ERNST GRELL
Max-Planck-Institut fur Biophysik
Heinrich-Hoffmann-StraBe 7
6000 Frankfurt 71
ISBN-13: 978-3-642-81539-3 e-ISBN-13: 978-3-642-81537-9
DOl: 10.1007/978-3-642-81537-9
Library of Congress Cataloging in Publication Data
Main entry under title: Membrane spectroscopy. (Molecular biology, biochemistry,
and biophysics; 31) 1. Membranes (Biology) 2. Spectroscopy. I. Grell, Ernst, 1941-
II. Series. [DNLM: 1. Membranes. 2. Spectrum analysis.
WI M0195T no. 31/ QH 601 M533) QH601.M4688 574.87'5'028 80-24524
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Softcover reprint ofthe hardcover 1s t edition 1981
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2131/3130-543210
Foreword
The last 10 years have seen an enormous growth in our understanding of the molecular
organisation of biological membranes. Experimental methods have been devised to meas
ure the translational and rotational mobility of lipids and proteins, thereby furnishing a
quantitative basis for the concept of membrane fluidity. Likewise, the asymmetry of bi
layer membranes as evidenced by the asymmetric insertion of proteins and lipids has been
put on firm experimental ground. At higher molecular resolution it has been possible to
provide a detailed pi2ture of the molecular conformation and dynamics of lipids and, to
some extent, even of small peptides embedded in a bilayer matrix. Many of these achieve
ments would not have been possible without the application of modem spectroscopic
methods. Since these techniques are scattered in a variety of specialized textbooks the
present monograph attempts to describe the key spectroscopic methods employed in
present-day membrane research at an intermediate level. There is no question that the
elusive detailed structure of the biological membrane demands a multiplicity of experi
mental approaches and that no single spectroscopic method can cover the full range of
physical phenomena encountered in a membrane. Much confusion in the literature has
arisen by undue generalizations without considering the frequency range or other limi
tations of the methods employed. It is to be hoped that the present monograph with its
comprehensive description of most modem spectroscopic techniques, will contribute to-
. wards a further convergence of views among the spectroscopic specialists and will enhance
the understanding of membrane structure.
Basel, September 1980 Joachim Seelig
Preface
The aim of this book is to introduce the reader to the application of spectroscopic tech
niques to the study of membranes. The following principal methods are covered: mag
netic resonance, optical and low-angle X-ray spectroscopy and chemical relaxation spec
trometry.
Each chapter summarizes the experimental and theoretical principles of a particular
technique and the special applications of that technique to the investigation of mem
branes. In addition, the contributions will critically review the current exploitation of
the technique, from the point of the view of the authors, by considering the results ob
tained on membrane constituents, simple model membranes and on biological membrane
systems of a highly complex nature. A common aspect in all the contributions is the in
tensive search for a detailed understanding of the structures and functions of biological
membranes at a molecular level.
This book will help to facilitate and to stimulate future studies in this interesting
field.
Frankfurt, Summer 1980 Ernst Grell
Table of Contents
1. Nuclear Magnetic Resonance Studies of the Phospholipid Bilayer
Membrane, S.1. Chan, D.F. Bocian, and N.O. Petersen (With 25 Figures) . 1
I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
II. Partially Oriented Molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 3
III. Effects of Motional Restriction on NMR Spectra. . . . . . . . . . . . . . . . . 6
IV. Isotropic Motion in the Presence of Restricted Local Motion:
Membrane Vesicles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 21
V. Membrane Dynamics from NMR Relaxation Measurements. . . . . . . . .. 23
VI. Merits of Various Spin Probes in Membrane Studies. . . . . . . . . . . . . .. 32
VII. Selected Applications ofNMR to Membrane Studies. . . . . . . . . . . . .. 38
VIII. Future Prospects. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 46
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 47
2. Electron Spin Resonance: Spin Labels, D.Marsh(With 33 Figures) . . . .. 51
I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 51
II. Experimental Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 53
III. Theoretical Basis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 67
IV. Applications to Membrane Studies. . . . . . . . . . . . . . . . . . . . . . . . .. 86
V. New Developl;l1ents and Future Trends ....................... 128
References .............................................. 137
3. Absorption and Circular Dichroism Spectroscopies, M.M. Long and
D.W. Urry (With 14 Figures) .............................. 143
I. Introduction ......................................... 143
II. Solution Studies ...................................... 143
III. Suspensions of Homogeneous Particles . . . . . . . . . . . . . . . . . . . . . . . 150
IV. Suspension of Biomembranes (Heterogeneous Particles) . . . . . . . . . . . . 154
References ...... ,. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170
4. Optical Spectroscopy of Monolayers, Multilayer Assemblies, and
Single Model Membranes, J. Heesemann and H.P. Zingsheim (With
6 Figures) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172
I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172
II. General Considerations and History . . . . . . . . . . . . . . . . . . . . . . . . . 173
x
Table of Contents
III. Design of Spectroscopic Probes ............................ 175
N. Concentration, Orientation, and Location of Chromophores . . . . . . . . . 178
V. Monomers, Dimers, and Aggregates .......................... 180
VI. Energy Transfer ...................................... 181
VIT. Lipoid pH-Indicators in Monolayers . . . . . . . . . . . . . . . . . . . . . . . . . 182
VIII. Lateral Diffusion in Model Membranes . . . . . . . . . . . . . . . . . . . . . . . 185
IX. Electric-Field-Induced Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194
s.
Fluorescence Spectroscopy of Biological Membranes, J. Yguerabide
and M.C. Foster (With 24 Figures) .......................... 199
I. Basic Theory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200
II. Types of Fluorophores Used in the Study of Membranes ............ 209
III. Applications ......................................... 214
References ............................................... 267
6. Infrared Membrane Spectroscopy, U.P. Fringeli and Hs.H. Gtinthard
(With 26 Figures) ..................................... 270
I. Introduction. ........................................ 270
II. Experimental Techniques 1 Spectrophotometric Consideration ....... 271
III. Application of IR Spectroscopy to Biological Systems ............. 272
N. Review of Results of IR Membrane Spectroscopy ................ 278
References .............................................. 327
7. Chemical Relaxation Spectrometry, H. Ruf and E. Grell (With 12 Figures) . 333
I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333
II. Experimental Methods .................................. 337
III. Theoretical Aspects .................................... 344
N. Applications to Membrane Studies .......................... 361
References .. : ........................................... 372
8. Raman Spectroscopy of Membrane Constituents and Related
Molecules, R.C. Lord and R. Mendelsohn (With 18 Figures) ......... 377
I. Introduction. ........................................ 377
II. Experimental Methods .................................. 383
III. The Theoretical Basis of Raman Spectroscopy .................. 389
N. Applications to Membrane Studies .......................... 397
V. Future Trends ........................................ 424
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 426
Table of Contents XI
9. Low-Angle X-Ray Diffraction, N.P. Franks and Y.K. Levine (With
23 Figures) ......................................... 437
I. Introduction ......................................... 437
II. Experimental Methods ....... 438
A ••••••••••••••••••••••••••
III. Theory............................................ 439
N. Applications to Membrane Systems ......................... 457
V. Conclusions ......................................... 482
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 483
Subject Index .............................................. 488
Nuclear Magnetic Resonance Studies of the Phospholipid
Bilayer Membrane
S.1. Chan, D.F. Bocian and N.O. Petersen
I. Introduction
The cell membrane has been the focus of much recent biochemical and biophysical re
search, primarily because of its role in cellular phenomena. Numerous efforts have been
directed toward determining the motional state of the lipid components of the mem
brane, the motivation being the contention that the lipid bilayer is the basic matrix in
which membrane proteins are embedded to form the biological membrane. As such, it is
likely that such diverse phenomena as maintenance of ionic gradients and transmembrane
potentials, activities of membrane-bound enzymes, interactions between membrane pro
teins, transmembrane signal transmission, intercellular communication, and manifesta
tions of cellular development and cell transformation all depend on the structure and
fluidity of the lipid bilayer.
The working hypothesis for the structure of the cell membrane is the fluid mosaic
model (Singer and Nicolson, 1972), where the steady state arrangements of lipids and
proteins are intrinsically dynamic in nature. Thus, the structural arrangement of mem
brane components contains essential information concerning membrane function. Simi
larly, understanding the dynamics of membrane components both in terms of internal
freedom of motion and in terms of lateral movements of the components in the lipid
. phase is important for revealing how the various time-dependent membrane functions
are controlled (Edidin, 1974). It is particularly in this latter area of membrane research
that various spectroscopic techniques such as magnetic resonance are useful.
Biological membranes are multicomponent systems composed of many different
membrane proteins and lipids. One of the primary lipid constituents of most biological
membranes)s phosph~lipids, which occur naturally with a variety of headgroups and
acyl chains. Aqueous dispersions of phospholipids exhibit many of the physical proper
ties of natural membranes, and accordingly these dispersions have been used extensively
as membrane models for magnetic resonance studies (Lee et al., 1974; James, 1975;
Tiddy, 1975, 1977; Seelig, 1977; Wennerstrom and Lindblom, 1977; Bocian and Chan,
1978). Phospholipids spontaneously aggregate to form bilayer structures, and the inter
actions between different bilayers result in a thermodynamically stable multilamellar
superstructure (Fig. 1).
Pure phospholipid dispersions exist in a number of phases depending on temperature
and water content. The two phases of primary importance as biological membrane mod
els are the gel and the liquid crystalline phases. In multicomponent phospholipid systems,
A.A. Noyes Laboratory of Chemical Physics (Contribution No. 5853), Californialnstitute of Tech
nology, Pasadena, California 91125, USA
2 S.1. Chan, D.F. Bocian and N.D. Petersen
Fig. 1. A representation of the muItiiamellar superstructure formed by phospholipid/water dispersions.
The individual lipid bilayers are of infinite extent and are separated by the interstitial water (Khetra
pal et aI., 1975)
species immiscibility and phase separations can occur and the phase diagrams become ex
ceedingly complicated. This phenomenon has been investigated extensively by electron
spin resonance (ESR) spin-label studies (Wu and McConnell, 1975). However, most nu
clear magnetic resonance (NMR) studies have been performed on single-component sys
tems and, in general, at temperatures above the gel ~ liquid crystalline phase transition
temperature. In principle, NMR could be used to study multicomponent systems if the
components could be suitably labeled. Also, sensitivity and abundance have made lipids
the principal membrane components investigated to date by NMR. However, the direct
observation of nonlipid membrane components is now becoming possible through the
use of specific labeling of these components, and by the use of lipid components which
do not contain the nucleus being observed (Feigenson et aI., 1977).
For many years, much of our knowledge about the motional state of the bilayer has
come from ESR measurements on spin-labeled lipids incorporated into the bilayer (Hub
bell and McConnell, 1971; Jost et aI., 1971; Schindler and Seelig, 1974). Recent efforts
have been directed toward developing spectroscopic probes which would circumvent the
possible perturbations in the bilayer produced by the bulky nitroxide radical. Nuclear
magnetic resonance spectroscopy is a natural tool for this purpose and there has been
considerable effort using lH, 2H, l3C, and 3lp probes which are either endogenous or in
corporated into the lipid molecule (Lee et aI., 1974; James, 1975; Tiddy, 1975, 1977;
Seelig, 1977; Wennerstrom and Lindblom, 1977; Bocian and Chan, 1978). Both ESR
